Transition state analysis of Vibrio cholerae sialidase-catalyzed hydrolyses of natural substrate analogues.

A series of isotopically labeled natural substrate analogues (phenyl 5-N-acetyl-α-d-neuraminyl-(2→3)-ß-d-galactopyranosyl-(1→4)-1-thio-ß-d-glucopyranoside; Neu5Acα2,3LacßSPh, and the corresponding 2→6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for Vibrio cholerae sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Acα2,3LacßSPh for the ring oxygen ((18)V/K), leaving group oxygen ((18)V/K), C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.029 ± 0.002, 0.983 ± 0.001, 1.034 ± 0.002, and 1.043 ± 0.002, respectively. In addition, the KIEs for Neu5Acα2,6ßSPh for C3-S deuterium ((D)V/K(S)) and C3-R deuterium ((D)V/K(R)) are 1.021 ± 0.001 and 1.049 ± 0.001, respectively. The glycosylation transition state structures for both Neu5Acα2,3LacßSPh and Neu5Acα2,6LacßSPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening ((4)H(5) half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Acα2,6ßSPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer.

Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus.

A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed beta-propeller structure with the model's highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl alpha-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl alpha-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred alpha2-3-linked sialic acids over the alpha2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes.

The hydrolase and transferase activity of an inverting mutant sialidase using non-natural beta-sialoside substrates.

The Y370G inverting mutant sialidase from Micromonospora viridifaciens possesses beta-sialidase activity with phenyl beta-sialoside (Ph-betaNeuAc) to give alpha-sialic acid as the first formed product. The derived catalytic rate constants for k(cat) and k(cat)/K(m) are 13.3 +/- 0.3 and (2.9 +/- 0.3) x 10(5) M(-)(1) s(-)(1), respectively. This enzyme is highly specific for the phenyl substrate, with substituted phenyl and thiophenyl leaving groups having k(cat) values that are at least 1000-fold lower. In addition, the Y370G mutant can transfer the sialic acid moiety from Ph-betaNeuAc to lactose in yields of up to 13%. Greater than 90% of the sialyl-lactose product formed in the coupling reactions is the alpha-2,6-isomer. A library encoding 6 x 10(5) different sialidases was constructed by mutating Y370, E260, T309, N310, and N311, residues that include and are proximal the catalytic tyrosine residue. A total of 2628 individuals were screened for hydrolytic activity against 4-nitrophenyl 2-thio-beta-sialoside and 4-methylumbelliferyl beta-sialoside. However, none of the mutants screened possessed a significant activity against either of the beta-sialosides.

Natural sialoside analogues for the determination of enzymatic rate constants.

Two isomeric 4-methylumbelliferyl-alpha-D-N-acetylneuraminylgalactopyranosides (1 and 2) were synthesised. These compounds contain either the natural alpha-2,3 or alpha-2,6 sialyl-galactosyl linkages, as well as an attached 4-methylumbelliferone for convenient detection of their hydrolyses. These compounds were designed as natural sialoside analogues to be used in a continuous assay of sialidase activity, where the sialidase-catalysed reaction is coupled with an exo-beta-galactosidase-catalysed hydrolysis of the released galactoside to give free 4-methylumbelliferone. The kinetic parameters for 1 and 2 were measured using the wild-type and nucleophilic mutant Y370G recombinant sialidase from Micromonospora viridifaciens. Kinetic parameters for these analogues measured using the new continuous assay were in good agreement with the parameters for the natural substrate, 3'-sialyl lactose. Given the selection of commercially available exo-beta-galactosidases that possess a variety of pH optima, this new method was used to characterise the full pH profile of the wild-type sialidase with the natural sialoside analogue 1. Thus, use of these new substrates 1 and 2 in a continuous assay mode, which can be detected by UV/Vis or fluorescence spectroscopy, makes characterisation of sialidase activity with natural sialoside linkages much more facile.